The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
Nucleic acid ligases belong to a class of enzymes that catalyze phosphodiester bond formation between adjacent 3′-hydroxyl and 5′-phosphoryl termini in nucleic acid (e.g., RNA or DNA) in the presence of a cofactor, such as ATP or NAD+. Ligases are employed in a number of molecular biology applications including nucleic acid sequence detection, single nucleotide polymorphism (SNP) detection, protein detection, sequencing by ligation, and ligase chain reaction (LCR).
In biochemical fidelity experiments, DNA ligases have been found to tolerate a variety of nucleic acid substrate mismatches. For example, T4 DNA ligase has a tolerance for mismatches that results in a propensity to seal one of every 103 mismatched duplexes. Showalter, A. K., et al., 106 Chem. Rev, 340-360 (2006). In comparison, the error rate of a conventional DNA polymerase is approximately one error for every 105-106 dNTP insertions, several orders of magnitude higher in fidelity than ligase. Other atypical joining reactions of DNA ligase include intramolecular loop formation (Western, L., et al., 19 Nucleic Acids Res, 809-813 (1991)), joining of non-overlapping, blunt-ended duplexes (Barringer, K., et al., 89 Gene, 117-122 (1990), Cao, W., 22 Trends Biotechnol., 38-44 (2004)) and template-independent reactions (Barringer, K., et al., Kuhn, H., et al., 272 FEBS J, 5991-6000 (2005)).
Various approaches have been described for improving DNA ligation fidelity. For example, Luo, J., et al., 24 Nucleic Acids Res, 3079-3085 (1996) disclose modifying the third nucleotide upstream from the 3′-OH, acceptor with universal base 3-nitropyrrole and site directed mutagenesis of the ligase protein. Tong, J., et al., 27 Nucleic Acids Res, 788-794 (1999); Feng, H., et al., 43 Biochemistry, 12648-12659 (2004); Jeon, H., et al., 237 FEMS Microbiol Lett., 111-118 (2004); Lim, J., et al., 388 Arch Biochem Biophys., 253-260 (2001); and Luo, J., et al., 24 Nucleic Acids Res, 3071-3078 (1996) disclose mutating amino acid residues in the DNA ligase. Cao, W., 22 Trends Biotechnol., 38-44 (2004) disclose using an endonuclease in the ligation reaction. Egholm, M., et al., U.S. Pat. No. 6,297,016 disclose acceptor modifications. Fung, S., et al., U.S. Pat. No. 5,593,826 discloses 3′-NH2 substituted acceptor probes. Bandaru, R., et al., U.S. Pat. Nos. 6,811,986 and 6,635,425 discloses use of 5′-thiophosphates in the donor (5′-phosphate) strand.
Modified ligase cofactors have been used determine ligase cofactor dependence and as ligation inhibitors. See e.g., Montecucco, A., et al., 271 Biochem J., 265-268 (1990); Shuman, S., 34 Biochemistry, 16138-16147 (1995); Raae, A., et al., 81 Biochem. Biophys. Res. Commun., 24-27 (1978); Cherepanov, A. V., et al., 269 Eur. J. Biochem., 5993-5999 (2002); Belford, H. G., et al., 268 J Biol Chem, 2444-2450 (1993); Doherty, A. J., et al., 271 J Biol Chem, 11083-11089 (1996); Ho, C. K., et al., 71 J Virol, 1931-1937 (1997); Lai, X., et al., 6 Extremophiles, 469-477 (2002); and Sriskanda, V., et al., 28 Nucleic Acids Res, 2221-2228 (2000).